Hapalindole U( 4)i savalidated biosynthetic precursor to ambiguine alkaloids (Angew.C hem. Int. Ed. 2016,55, 5780), of whichbiogenetic origin remains unknown. The recent discovery of AmbU4 (or FamC1) protein encoded in the ambiguine biosynthetic pathway (J.A m. Chem. Soc. 2015,1 37, 15366), an isomerocyclase that can rearrange and cyclizegeranylated indolenine (2)toapreviously unknown 12epi-hapalindole U(3), raised the question whether 3 is adirect precursor to 4 or an artifact arising from the limited in vitro experiments.Here we report asystematic approachthat led to the discovery of an unprecedented calcium-dependent AmbU1-AmbU4 enzymatic complex for the selective formation of 4.T his discovery refuted the intermediacy of 3 and bridged the missing links in the early-stage biosynthesis of ambiguines.This work further established the isomerocyclases involved in the biogenesis of hapalindole-type alkaloids as an ew family of calcium-dependent enzymes,w here the metal ions are shown critical for their enzymatic activities and selectivities.

The cyanobacterial prenyltransferase AmbP3 catalyzes the reverse prenylation of the tetracyclic indole alkaloid hapalindole U at its C‐2 position. Interestingly, AmbP3 also accepts hapalindole A, a halogenated C‐10 epimer of hapalindole U, and catalyzes normal prenylation at its C‐2 position. The comparison of the two ternary crystal structures, AmbP3‐DMSPP/hapalindole U and AmbP3‐DMSPP/hapalindole A, at 1.65–2.00 Å resolution revealed two distinct orientations for the substrate binding that define reverse or normal prenylation. The tolerance of the enzyme for these altered orientations is attributed to the hydrophobicity of the substrate binding pocket and the plasticity of the amino acids surrounding the allyl group of the prenyl donor. This is the first study to provide the intimate structural basis for the normal and reverse prenylations catalyzed by a single enzyme, and it offers novel insight into the engineered biosynthesis of prenylated natural products.